The adult mammalian heart shows little or no significant natural regeneration of cardiac muscle after injury, and instead heals by scarring. This regenerative deficiency has enormous socioeconomic consequences, given that ischemic myocardial infarction is a leading cause of morbidity and mortality in the United States and over 5 million Americans suffer from heart failure. Many years ago, we found that the teleost zebrafish displays a robust regenerative response after partial resection of the cardiac ventricle, involving creation of new cardiomyocytes with little or no scarring. Pre-existing cardiomyocytes, not stem cells, are the primary source of new muscle during heart regeneration in zebrafish. Muscle regeneration is influenced by activities of the epicardium and endocardium, major non-muscle cells that line the cardiac chambers. Progress during the previous periods of this grant has opened several avenues of investigation to address hurdles to how and why heart regeneration occurs. Most importantly, we have generated new strategies and tools for exploring the gene regulatory mechanisms of heart regeneration, and to determine functions and mechanisms of secreted and systemic factors that activate cardiomyocyte proliferation in response to injury. The goals of this application are to address central questions about the regulation of bona fide heart regeneration, using cutting edge screening and analysis tools in zebrafish. 1) We will define chromatin regulatory signatures and enhancer elements for heart regeneration, using genome-wide chromatin structure profiles obtained from regenerating cardiac tissue. 2) We will use new tools to define responses in distant tissues that potentially impact heart regeneration. 3) We will define functions of new modulators of heart regeneration identified from an in vivo screening strategy that visualizes proliferating cardiomyocytes in live zebrafish. With this approach, we will test the hypothesis that local and distant injury responses control a mitogenic regulatory program for heart regeneration. Our work will provide a detailed understanding of cardiac regeneration and identify key regulators. These discoveries will inform approaches for comprehending and enhancing the limited regenerative response displayed by humans after myocardial infarction.